The DAPI was excited by the 355nm laser and its emission collected using a 450/50 filter. Preparation and sequencing of RNA-seq libraries RNA prepared from flow sorted cells was quality controlled on a Bioanalyzer 2100 (Agilent) using RNA Pico 6000 chips (Agilent 5067C1513). for 3 minutes at 4C before RNA extraction with TRI Reagent. 20% of RNA obtained was separated on a 1.2% glyoxal gel and imaged by ethidium bromide staining. B: COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 100% methanol on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x for 3 minutes at 4C before RNA extraction with TRI Reagent. RNA was analysed as in A. C: COLO205 cells were fixed with glyoxal fixation mix (pH4) either without or with 20% ethanol and incubated on ice for 15 minutes and washed once in PBS by centrifugation at 2000 x for 3 minutes at 4C. RNA was extracted and analysed as in A. D: COLO205 cells were either unfixed (lane 1), fixed with glyoxal fixation mix (pH4) with 20% ethanol (lane 2) or with 4% formaldehyde on ice for 15 minutes (lane 3). Cells were washed once in PBS by centrifugation at 2000 x for 3 minutes at 4C and incubated on ice for 1 hour in 100 l PBS followed by centrifugation at 2000 x for 3 minutes at 4C before RNA extraction with an RNeasy mini kit. E: 100 ng RNA per reaction from D was subjected to one-step combined reverse transcription and quantitative PCR reactions for and along with many other known cell-cycle regulated transcripts (S2 Table). Unsurprisingly, GO enrichment analysis for these transcripts identifies “mitotic cell cycle process” as the most enriched category (FDR corrected q-value 2.7×10-65) along with ~300 other significant enrichments (q 0.05) almost all of which are directly connected to cell cycle progression (S3 Table). Overall, these data show that cells can be stained for an intracellular antigen and accurately sorted using our glyoxal fixation protocol, yielding high-quality, largely unbiased RNA samples that give rise to high quality RNA-seq libraries. Discussion Although formaldehyde and glyoxal can be utilised similarly in both protein fixation and RNA denaturation, they react very differently with mixed RNA-protein substrates. This chemical difference gave rise to our prediction that glyoxal should fix cell samples without permanently modifying or crosslinking RNA. Here we have confirmed that RNA remains intact and accessible after glyoxal fixation, even through extended staining and sorting procedures. We further show that mRNA recovered from glyoxal-fixed cells can be purified by hybridisation to oligo(dT) and can be efficiently reverse transcribed. Glyoxal fixative is easy to prepare and use, and should be straightforward to substitute for formaldehyde in most protocols. However we find Typhaneoside that staining protocols do need to be modified to maintain RNA quality: firstly, inclusion of RNasin or an equivalent placental RNase inhibitor proved to be important. Secondly, we performed antibody incubations on ice to further reduce RNase activity, though we suspect this is not absolutely required. Thirdly, some staining reagents are clearly incompatible with RNA isolationharsh detergents such as Triton X-100 allow RNA to escape the cell (an unavoidable consequence of the RNA not being crosslinked), and serum can contain RNase Typhaneoside although RNA compatible blocking agents are commercially available [8]. We found it beneficial to perform trial stainings on the sample-type of interest and assess RNA quality by Bioanalyzer or RNA mini-gel (for which we provide a simple and robust Typhaneoside Typhaneoside protocol in Materials and Methods) as well as confirming staining quality before attempting a cell sorting experiment. It should however be noted Nrp2 that we did not need to render bulk staining solutions RNase free (e.g.: using DEPC), nor did we undertake any special cleaning of the flow cytometer or use special sheath fluid that would make Typhaneoside routine use of these methods more arduous. Whether glyoxal is a better or worse fixative than formaldehyde for microscopy studies of protein antigens remains a matter of dispute, and likely varies depending on precise cell type and target [30, 36]. In contrast, our data confirm that glyoxal is a far superior fixative for RNA applications because RNA remains extractable and not permanently modified in fixed cell samples. This means that substitution of formaldehyde with glyoxal and minor adjustments to staining buffers should be sufficient to render standard cell staining and sorting protocols compatible with a wide range of RNA methods including bulk and single cell RNA-seq. Materials and methods Step-by-step up-to-date protocols describing these methods are available from the JH group website at https://www.babraham.ac.uk/our-research/epigenetics/jon-houseley/protocols Cells and cell culture COLO205 and MCF7 cell lines were provided by the laboratory of Dr. Simon J Cook at the Babraham Institute. All cell culture reagents.
